Resistance of pancreatic cancer to current treatment protocols as a consequence of apoptosis defects is a major cause of treatment failure and poor prognosis. Therapeutic approaches that specifically target components of these molecular mechanisms are urgently needed. The anti-apoptotic myeloid cell leukemia protein Mcl-1, a member of the Bcl-2 family proteins, has emerged as a promising therapeutic target. Consistent with its anti-apoptotic function, the overexpression of Mcl-1 in pancreatic cancer cells has been associated with tumor progression and resistance to current chemotherapeutics. It was demonstrated that Mcl- 1 is an important survival factor for pancreatic cancer cells; its down-regulation with siRNA for example, enhances the induction of apoptosis, chemosensitivity and radiosensitivity of pancreatic cancer cells. There are currently no known specific small-molecule inhibitors of Mcl-1 and targeting Mcl-1 to overcome apoptosis resistance is an important strategy for the development of new drugs to treat pancreatic cancer. For this R21 award we propose development of small-molecule Mcl-1 inhibitors as a new targeted therapy for treatment of pancreatic cancer. We hypothesize that Mcl-1 inhibitors which can selectively block its interaction with the pro-apoptotic proteins, Bak and Bax, will inhibit Mcl-1's anti-apoptotic functions, induce apoptosis and inhibit the growth of pancreatic cancer cells. We further posit that inhibition of Mcl-1 function will enhance the response to chemotherapeutics currently used in the treatment of human pancreatic cancer. Through the NIH MLSCN Program and in collaboration with the Chemical Biology Discovery Center at Emory University, we have applied a high throughput screening (HTS) approach to discover small-molecule Mcl-1 inhibitors. We have performed extensive counter screens and functional characterization in order to validate the most promising cluster, the E cluster. We have designed and synthesized several analogues and the most potent compound, E288, has 13 to 6-fold improved binding affinity to Mcl-1 protein than identified hits (Ki = 400 nM). This compound shows selectivity over Bcl-2 and Bcl-xL. NMR spectroscopy demonstrates that E288 binds to the same BH3 domain of Mcl-1 as the Bim BH3 peptide and antagonizes Mcl-1, inhibiting cell growth and inducing apoptosis in pancreatic cancer cells with high Mcl-1 levels. Preliminary mechanisms of action studies indicate that these inhibitors induce apoptosis in time and dose-dependent manners and that the pro-apoptotic proteins, Bax and Bak are involved in the induction of apoptosis. We propose further optimization of E288 through a structure-based strategy, integrating computer modeling, design and synthesis, and extensive in vitro and in vivo evaluation of the activity of the most potent Mcl-1 inhibitors for their potential, alone or in combination in the treatment of pancreatic cancer. Successful completion of the study should provide unambiguous data on the therapeutic potential of Mcl-1 inhibitors against human pancreatic cancer and gain important insights into the molecular details of this approach. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is a lethal disease whose treatment has largely been unsuccessful because pancreatic tumor cells are resistant to standard chemotherapy. A major regulator of apoptosis, the Mcl-1 protein, is highly up-regulated in pancreatic cancer cells and is responsible for resistance to chemotherapeutic agents. To develop Mcl-1 inhibitors for the treatment of pancreatic cancer in which Mcl-1 is overexpressed, in this application we propose further optimization of our new discovered small-molecule Mcl-1 inhibitors and extensive in vitro and in vivo evaluation of their anti-tumor activity alone or in combination with standard chemotherapeutic agents.